Bacteria are able to sense and respond to their environment through multi-protein signal transduction cascades, the most common of which is the histidine-aspartate sensory pathways (HAPs). The most characterized HAP is a bacterial motility system termed bacterial chemotaxis. The components of the chemotaxis core signaling complex are conserved among motile bacteria and within HAPs. This research will investigate fundamental principles underlying bacterial signal transduction mechanisms using Escherichia coli as a model system. Despite over five decades of research and extensive characterization of the individual E. coli chemotaxis pathway components, a full molecular level understanding of signal transduction has not been elucidated. Signal transduction in chemotaxis is initiated by the binding of extracellular ligands to a specialized family of transmembrane receptors. These transmembrane receptors or chemoreceptors, cluster at distinct regions of the cell and are arranged in an extended lattice. Chemoreceptor organization is conserved across bacteria. However, the importance of this organization has yet to be fully realized. The application of multivalent ligands to the chemotaxis system afforded the first evidence that an extended, membrane associated lattice of chemotaxis signaling proteins is critical for transducing signals. Additionally, our group provided evidence that attractants transduce signals by disrupting organization within the signaling array by demonstrating receptor delocalization upon activation by immuno-fluorescence microscopy. This formulated a hypothesis that changes in the conserved array organization controls signaling. We aim to image changes in chemoreceptor organization upon stimulation. Chemoreceptor imaging will be performed by electron cryotomography (ECT); an electron microscopy technique that allows for 3D reconstructions of nanometer scale biological structures. Rapid freezing of samples permits visualization of preserved protein organizations. Advances within our group developing ligand polymers will provide the tools necessary to probe receptor organization. The synthetic tractability of these polymers will allow the appendage of a fluorophore for imaging by combination fluorescent/ECT providing assurance that images are of actively engaged receptors. The results of this work will be vital to understanding bacterial motility and transmembrane signaling in general. The proposed tools for ECT will be broadly applicable to elucidating features of other important biological systems. Moreover, the chemotaxis system has been implicated in regulating the differentiation of some bacteria to a pathogenic swarmer cell state. Uncovering the chemotaxis signaling mechanisms will have ramifications in understanding the impact of the chemotaxis system on bacterial pathogenicity and provide a new unexplored mode for control and mitigation.